Modified defensins and their use

ABSTRACT

This disclosure provides modified antimicrobial agents, for example modified defensin polypeptides. Compositions including a modified arginine residue, such as an ADP-ribosylated and/or ribosylated alpha defensin polypeptide, are provided. Also provided are methods of modulating an immune response using the modified defensin polypeptides. Methods are provided for modulating an antimicrobial activity and for inhibiting a cytotoxic activity. Also disclosed are methods for treating diseases in a subject that are associated with an immune response, such as inflammatory and pulmonary diseases, using the disclosed modified defensin polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 10/504,838, whichwas filed Aug. 13, 2004, and met the requirements under 35 U.S.C. §371(c)(1), (c)(2), and (c)(4) on Jun. 23, 2005, now U.S. Pat. No.7,511,015, issued on Mar. 31, 2009, which is the §371 U.S. NationalStage of International Application No. PCT/US03/04649, filed Feb. 18,2003, which was published in English under PCT Article 21(2), which inturn claims the benefit of U.S. Provisional Application No. 60/358,504,filed Feb. 19, 2002, all of which are incorporated by reference hereinin their entirety.

FIELD

The present disclosure relates generally to antimicrobial agents, suchas defensins, and, more specifically, to a modified defensins such asadenosine-diphosphate (ADP)-ribosylated defensins and their use inmodulating an immune response.

BACKGROUND

Defensins are small, cationic peptides containing six conserved cysteineresidues that form three disulfide bonds. Functional, mature defensinsarise by the sequential post-translational processing of prepro-proteinsthat are 93-95 amino acids in length. Mature alpha-defensins generallycontain 29-33 residues, whereas mature beta-defensins are more basic andare generally between 34-37 amino acids in length. The recentlyidentified theta defensins are formed by the head-to-tail ligation oftwo alpha defensin-related nonapeptides, generating a circular18-residue polypeptide.

Defensins were first identified in neutrophils and have been detected inhuman, rabbit, guinea pig, and rat phagocytes. Four human alphadefensins have been isolated from neutrophils: human neutrophil peptide(HNP)-1, HNP-2, HNP-3, and HNP-4. HNP-1, HNP-2, and HNP-3 are stored inthe azurophilic granules of neutrophils and constitute approximately 99%of the defensin content of the neutrophils. HNP-4 is also present inthese granules, but at a concentration that is equivalent to only onepercent of the other HNP polypeptides. Alpha defensins also include twohuman enteric defensins, human defensin (HD)-5 and HD-6, which arehighly expressed in epithelial cells of the small intestine,specifically in the Paneth cells.

Defensins play important roles in the innate immune defense invertebrates. Defensins are broad-spectrum antimicrobial molecules thatare released from azurophilic granules into a phagosome for thenonoxidative killing of phagocytized infectious agents such asGram-negative bacteria, Gram-positive bacteria, fungi, and certainenveloped viruses, by forming pores in their membranes. Constitutivelyexpressed defensins contribute to an antimicrobial barrier at theepithelial cell surface and inducible epithelial defensins are highlyexpressed at areas of inflammation or infection. Thus, defensins play animportant role in the body's natural immunity against infections.Defensins also play a role in the body's natural immunity against tumorcells.

The ubiquitous use of antibiotics has resulted in the selection ofbacteria that are relatively resistant to these drugs. Furthermore, fewdrugs are effective against viral and fungal microorganisms. Thus, thereis a continuing need to identify novel agents that reduce or inhibit thegrowth of such microorganisms as well as novel agents that may recruitinflammatory cells to enhance an immune response. Defensins exhibit awide range of antimicrobial activities, including cytotoxicity towardsbacterial cells, however these proteins are also cytotoxic for mammaliancells, including human epithelial and endothelial cells. This sideeffect may limit their usefulness as antimicrobial agents. Thus, therealso exists a need to identify new methods of modifying existing agents,such as defensins, in order to modify their cytotoxic activity and givethem superior antimicrobial activities.

SUMMARY

The disclosure provides a composition including a modified defensinpolypeptide. In one embodiment, an arginine residue of the defensinpolypeptide is modified by ADP-ribosylation or ribosylation to form anADP-ribosylated or ribosylated defensin. In one embodiment, the defensinpolypeptide is an alpha defensin. In another embodiment, the alphadefensin polypeptide is human neutrophil peptide-1. In one embodiment,the ADP-ribosylated or ribosylated defensin has antimicrobial activityless than that of unmodified defensin. In another embodiment, apharmaceutical composition is provided that includes a modified defensinpolypeptide.

Also provided herein is a substantially purified modified defensinpolypeptide, or a functional fragment of the polypeptide, wherein thedefensin or functional fragment is a cationic, arginine-rich polypeptidethat has an antimicrobial activity. In one embodiment, the defensinpolypeptide, or a functional fragment of the peptide, has at least onearginine residue that is ribosylated or ADP-ribosylated. In anotherembodiment, the defensin peptide, or a functional fragment of thepeptide has antimicrobial activity.

The disclosure also provides a method for increasing an antimicrobialactivity of a defensin polypeptide. Examples of these methods includeribosylating or ADP-ribosylating at least one arginine residue of thedefensin polypeptide in order to increase the antimicrobial activity ofthe defensin polypeptide.

Also provided herein is a method of modulating an immune responseagainst a microbe in a subject. The method includes administering to thesubject a therapeutically effective amount of a modified defensinpolypeptide. In one embodiment, the defensin polypeptide has anADP-ribosylated or ribosylated arginine residue.

Also disclosed is a method of inhibiting a cytotoxic activity of anon-ADP-ribosylated or a non-ribosylated defensin polypeptide in asubject. In one embodiment, the method includes administering to thesubject a therapeutically effective amount of an ADP-ribosylated or aribosylated defensin in order to inhibit a cytotoxic activity of thenon-ADP-ribosylated or non-ribosylated defensin.

A method is also disclosed for treating a subject who has a pulmonarydisease. The method includes administering to the subject atherapeutically effective amount of a modified defensin polypeptide inorder to treat the pulmonary disease. In one embodiment, an arginineresidue in the defensin polypeptide is modified by ADP-ribosylation orribosylation.

Also provided herein is a method for screening for an agent that affectsADP-ribosylation or ribosylation of a defensin. The method includescontacting a cell that expresses an ADP-ribosyltransferase with theagent and the defensin polypeptide, and evaluating the ADP-ribosylation.The method also includes contacting a cell that expressesADP-ribosyltransferase and pyrophosphatase/phosphatase with the agentand the defensin polypeptide, and evaluating the ribosylation of thedefensin polypeptide.

The disclosure also provides a pharmaceutical composition comprising atherapeutically effective amount of a ribosylated antimicrobial agent oran ADP-ribosylated antimicrobial agent in a pharmaceutically acceptablecarrier. Specific, non-limiting examples of an antimicrobial agentinclude a defensin polypeptide, such as an alpha defensin, for examplehuman neutrophil peptide-1, or lysozyme.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a series of reverse-phase high-performance liquidchromatography (RP-HPLC) elution profiles that demonstrate theseparation of the products generated by ADP-ribosyltransferase(ART)-1-catalyzed ADP-ribosylation of HNP-1. HNP-1 was incubated withreaction buffer alone (FIG. 1A), nicotinamide adenine dinucleotide(NAD⁺) (FIG. 1B), ART-1 (FIG. 1C), or ART-1 plus NAD⁺ (FIG. 1D) and thenanalyzed by RP-HPLC. Absorbance at 210 nm, is shown as a function ofelution time. Full scale for FIG. 1A and FIG. 1B is 750 units, for FIG.1C is 650 units and for FIG. 1D is 250 units. ADP-ribosylated HNP-1 andHNP-1 are indicated by solid and open arrows, respectively. Absorbancespectra of ADP-ribosylated HNP-1 (solid line) and HNP-1 (dotted line)were obtained during elution using an inline array detector (inset).

FIG. 2 is a series of dose curves that demonstrate the antimicrobial andcytotoxic activities of ADP-ribosylated HNP-1 and non-ADP-ribosylatedHNP-1. FIG. 2A is a graph demonstrating the cytotoxic activity ofADP-ribosylated HNP-1 versus that of non-ADP-ribosylated HNP-1 in aradial diffusion assay. Antimicrobial activity is positively correlatedwith the diameter of a zone cleared of E. coli growth by the indicatedconcentration of HNP-1 (◯) or ADP-ribosylated-HNP-1 (□), minus thediameter of the central well (3 mm). Data are means±½ range of valuesfrom 2 independent experiments. FIG. 2B is a graph demonstrating thecytotoxic activity of ADP-ribosylated HNP-1 versus that ofnon-ADP-ribosylated HNP-1 using a chromium release assay. Cytotoxicityis positively correlated with lysis of chromium (⁵¹Cr)-labeled A549cells during incubation with the indicated concentrations of HNP-1 (◯),ADP-ribosylated HNP-1 (□), or synthetic HNP-1 (sHNP) (Δ). Cell lysis ismeasured by the amount of chromium released from the damaged or brokencells. Percent of lysis was calculated as(cpm_(exp)−cpm_(spont))/(cpm_(max)−cpm_(spont))×100. Data aremeans±standard error of the mean (SEM) of values from 4 independentexperiments.

FIG. 3 is a series of dose curves that demonstrate the effects ofADP-ribosylated-HNP-1 on HNP-1 antimicrobial and cytotoxic activity.FIG. 3A is a graph demonstrating the effect of incubating HNP-1 (100 nM)with the indicated concentrations of ADP-ribosylated HNP-1 before it isadded to E. coli in a radial diffusion assay. Difference in zone size(♦) was calculated as in FIG. 2A. Data are means±SEM of values from 3independent experiments. FIG. 3B is a line graph demonstrating theeffect of incubating HNP-1 with ADP-ribosylated HNP-1 before it is addedto ⁵¹Cr-labeled A549 cells in a chromium release assay. HNP-1 (12 μM)was incubated with the indicated concentrations of ADP-ribosylatedHNP-1. Percentage of cell lysis (♦) was determined, as in FIG. 2B.Cytotoxicity is positively correlated with lysis of ⁵¹Cr-labeled A549cells. The cytotoxic effects of HNP-1 (◯) or ADP-ribosylated HNP-1 (□)are also shown. Data are means±½ range of values from 2 independentexperiments.

FIG. 4 is a series of bar graphs that demonstrate the effects ofADP-ribosylated HNP-1 and HNP-1 on interleukin (IL)-8 release by A549cells and on T cell chemotaxis. Cells were incubated for 12 hours (FIG.4A) or 24 hours (FIG. 4B) with the indicated concentrations of HNP-1 orADP-ribosylated HNP-1 before analysis of the media. The IL-8 present inthe medium of the cells incubated without defensins has been subtracted.Data are means±½ range of 2 separate experiments, each performed intriplicate. *P<0.05 for the difference in IL-8 release by HNP-1 andADP-R HNP-1. In FIG. 4C, CD3⁺ cells were incubated with 0.025, 0.25,2.5, or 25 nM of HNP-1 or ADP-ribosyl HNP-1. Migration medium withMacrophage Inflammatory Peptide (MIP)-1β (5 ng/ml) and migration mediumalone, were used as positive and negative controls, respectively.Chemotaxis percentage was calculated as follows: (number of cellsmigrated to the lower chamber in the experimental conditions−number ofcells migrated in the negative control)/(number of cells migrated in thepositive control−number of cells migrated in negative control)×100. Dataare shown as means±SEM of 4 separated experiments, each performed induplicate.

FIG. 5 is a series of elution profiles that characterize theADP-ribosylated defensins obtained from bronchoalveolar lavage fluid.FIG. 5A demonstrates the alignment of RP-HPLC chromatograms ofART-1-catalyzed ADP-ribosylation of HNP-1 (continuous line) and thebronchial lavage sample from a smoker (dotted line). Arrows indicateelution times of ADP-ribosylated HNP-1 (46.5 minutes) and HNP-1 (48.5minutes). In FIG. 5B profile 1 has a peak with an elution timecompatible with ADP-ribosylated HNP-1 (46.5 minutes) and shows a mass of3,983 daltons when analyzed by MALDI mass spectroscopy, consistent withADP-ribosylated HNP-1. Incubation of ADP-ribosylated HNP-1 withpyrophosphatase/phosphatase produced ribosyl-HNP-1 (calculated 3,574daltons) (profile 2) and incubation of ADP-ribosylated HNP-1 withADP-ribosylarginine hydrolase cleaved the ribose-arginine linkage torelease HNP-1 (calculated 3,443 daltons) (profile 3). The x axisrepresents mass to charge ratio (m/z).

FIG. 6 is a digital image of an autoradiogram. The lane corresponding tothe [³²P]NAD reaction mixture containing both ART-1 and 5 μg of lysozymereveals a strong band at approximately 14 kDa, consistent with[³²P]ADP-ribosylation of lysozyme.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

SEQ ID NO:1 is the amino acid sequence of the HNP-1, HNP-2, HNP-3prepro-protein.

SEQ ID NO:2 is the amino acid sequence of HNP-1.

SEQ ID NO:3 is the amino acid sequence of HNP-2.

SEQ ID NO:4 is the amino acid sequence of HNP-3.

SEQ ID NO:5 is the amino acid sequence of the HNP-4 prepro-protein.

SEQ ID NO:6 is the amino acid sequence of HNP-4.

SEQ ID NO:7 is the amino acid sequence of the HD-5 prepro-protein.

SEQ ID NO:8 is the amino acid sequence of HD-5.

SEQ ID NO:9 is the amino acid sequence of the HD-6 prepro-protein.

SEQ ID NO:10 is the amino acid sequence of HD-6.

SEQ ID NO:11 is the forward primer for mono-ADP-ribosyltransferase(ART)-1.

SEQ ID NO:12 is the reverse primer for ART-1.

SEQ ID NO:13 is the amino acid sequence of the Def-X prepro-protein

SEQ ID NO:14 is the amino acid sequence of Def-X.

DETAILED DESCRIPTION

I. Abbreviations

-   ADP adenosine-diphosphate-   ART ADP-ribosyltransferase-   ELISA enzyme-linked immunosorbent assay-   exp experimental conditions-   GPI glycosylphosphatidylinositol-   HD human defensin-   HNP human neutrophil peptide-   IL interleukin-   MALDI-MS Matrix Assisted Laser Desorption Ionization-Time of    Flight-Mass Spectrometry-   MIP-1β macrophage inflammatory protein-1β-   max maximal release-   NAD nicotinamide adenine dinucleotide-   RP-HPLC reverse-phase high-performance liquid chromatography-   SEM standard error of the mean-   spont spontaneous release    II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of theinvention, the following explanations of specific terms are provided:

ADP-ribosylation: Reaction in which ADP-ribose is covalently attached toanother compound. A family of eukaryotic and prokaryoticmono-ADP-ribosyltransferases (ARTs) catalyze the transfer of ADP-ribosefrom nicotinamide adenine dinucleotide (NAD) to the guanidino group ofarginine residues on proteins. Bacterial products (e.g., cholera toxin,pertussis toxin, diphtheria toxin) are included among the ARTs.

A family of mammalian ADP-ribosyltransferases that are secreted orlocalized on the cell surface through glycosylphosphatidylinositol (GPI)anchors are expressed preferentially on epithelial and inflammatorycells such as lymphocytes and neutrophils. Substrates of the five knownmammalian ADP-ribosyltransferases (ART-1, ART-2, ART-3, ART-4, ART-5)include proteins that are involved in critical cellular events (e.g.,lymphocyte activation, neutrophil chemotaxis). Two of thesetransferases, ART-1 and ART-5, specifically modify arginine residues inproteins.

As disclosed herein, a number of proteins used in host defense are basicand arginine-rich and thus could serve as acceptors for ADP-ribose.These include, but may not be limited to, alpha defensins (HNP-1, HNP-2,HNP-3, HNP-4, HD-5, HD-6); beta defensins (hBD1, hBD-2, hBD-3, hBD-4);Major Basic Protein; Eosinophil Cationic Protein; Human Cathelicin LL-37(hCAP18) and lysozyme.

Adult respiratory distress syndrome (ARDS): Sudden pulmonaryinterstitial and alveolar edema, which usually develops within a fewdays after an initiating trauma. ARDS is thought to result from alveolarinjury that has led to increased capillary permeability. Also calledacute respiratory distress syndrome.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antimicrobial agent: A substance, including, but not limited to, achemical compound, small molecule, peptide mimetic, peptide, or proteinfor killing microorganisms or suppressing their multiplication orgrowth. Specific, non-limiting examples of agents with antimicrobialactivity include, but are not limited to, defensins, lysozyme,myeloperoxidase, and bacterial/permeability-increasing protein (BPI). Anagent has “antimicrobial activity” if it can damage a microorganism insuch a way that it results in the death of a microorganism or suppressesthe growth of a microorganism. An antimicrobial activity includes, butmay not be limited to, cell lysis due to cytotoxicity. Antimicrobialactivity can result from T cell chemotaxis or neutrophil recruitment. Inone embodiment, an antimicrobial activity is the lysis of a bacterialcell.

Arginine: An amino acid (C₆H₁₄N₄O₂) found in plants and animals that isessential for the human diet; also produced by the breakdown ofproteins. Also encompassed are functional analogues of arginine, andstructurally modified arginine molecules (e.g., agmatine).

Asthma: A disorder of the respiratory system characterized byinflammation, narrowing of the airways and increased reactivity of theairways to inhaled agents. Asthma is frequently, although notexclusively, associated with atopic or allergic symptoms.

Bacterial/Permeability-Increasing Protein (BPI): Cationic proteinpresent in the azurophilic granules of polymorphonuclear leukocytes(PMN). BPI is toxic only toward Gram-negative bacteria. This specificityis attributable to the strong attraction of BPI for thelipopolysaccharides in the bacterial envelope.

B cell or B lymphocyte: One of the two major types of lymphocytes. Theantigen receptor on B lymphocytes, sometimes called the B cell receptor,is a cell-surface immunoglobulin. On activation by an antigen, B cellsdifferentiate into cells producing antibody molecules of the sameantigen-specificity as this receptor.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and regulatory sequences that determinetranscription. cDNA is synthesized in the laboratory by reversetranscription from messenger RNA extracted from cells.

Chronic Bronchitis: An inflammation of the lining of the bronchialtubes. When the bronchi are inflamed and/or infected, less air is ableto flow to and from the lungs and a heavy mucus or phlegm is coughed up,resulting in bronchitis. A brief attack of acute bronchitis with coughand mucus production can occur with severe colds. Chronic bronchitis ischaracterized by the presence of a mucus-producing cough most days ofthe month, three months of a year for two successive years without otherunderlying disease to explain the cough. It may precede or accompanypulmonary emphysema. Cigarette smoking is by far the most common causeof chronic bronchitis. The bronchial tubes of people with chronicbronchitis may also have been irritated initially by bacterial or viralinfections. Air pollution and industrial dusts and fumes are alsocauses. Once the bronchial tubes have been irritated over a long periodof time, excessive mucus is produced constantly, the lining of thebronchial tubes becomes thickened, an irritating cough develops, airflow may be hampered, and the lungs are endangered. The bronchial tubesthen make an ideal breeding place for infectious agents.

Crohn's Disease: An Inflammatory Bowel Disease, the general name fordiseases that cause inflammation in the intestines. Crohn's Diseasecauses inflammation in the small intestine. Crohn's Disease usuallyoccurs in the lower part of the small intestine (ileum) but it canaffect any part of the digestive tract, from the mouth to the anus. Theinflammation extends deep into the lining of the affected organ. Theinflammation can cause pain and can make the intestines emptyfrequently, resulting in diarrhea. Crohn's Disease may also be calledileitis or enteritis.

Chronic Obstructive Pulmonary Disease (COPD): Includes emphysema andchronic bronchitis—diseases that are characterized by obstruction to airflow. Emphysema and chronic bronchitis frequently coexist. It does notinclude other obstructive diseases such as asthma.

Cystic fibrosis: A recessive genetic disease in which the exocrineglands of afflicted individuals produce abnormally thick mucus whichblock the intestines and lung passageways and produce scarring andlesions in the lungs and pancreas.

Cytokines: Proteins made by cells that affect the behavior of othercells, such as lymphocytes and neutrophils. In one embodiment, acytokine is a chemokine, a molecule that affects cellular trafficking.Cytokines include, but may not be limited to, MIP-β, IL-1, IL-8, IL-10,granulocyte-macrophage colony stimulating factor (GMCSF), granulocytecolony stimulating factor (GCSF), neurokinin, and tumor necrosisfactor-alpha (TNF-α).

Defensins: Small, cationic peptides that have six conserved cysteineresidues that form three disulfide bonds. Functional defensins arise bythe sequential post-translational processing of a prepro-protein of93-95 amino acids in length. The members of the defensin family aredivided into different classes. The alpha-defensins generally contain29-33 residues. The beta-defensins are more basic than alpha defensinsand are generally between 34-37 amino acids in length (Raj et al.,Biochem J; 347:633-41, 2000). The recently identified theta defensinsare formed by the head-to-tail linkage of two alpha defensin-relatednonapeptides, generating a circular 18-residue polypeptide (Tang et al.,Science; 286:498-502, 1999).

Defensins were first identified in neutrophils and have been detected inhuman, rabbit, guinea pig, and rat phagocytes. Alpha defensins include,but are not be limited to, HNP-1, HNP-2, HNP-3, HNP-4, human defensin(HD)-5, and HD-6. Alpha defensins also include the recently identifiedHNP-4 homolog, defensin (Def)-X (see U.S. Pat. No. 6,329,340 hereinincorporated by reference). HNP-1, HNP-2, and HNP-3 are products of thesame gene (GenBank Accession No. P11479 herein incorporated byreference). HNP-4 is the product of a different gene (GenBank AccessionNo. NP_(—)001916 herein incorporated by reference). HD-5 (GenBankAccession No. NP_(—)066290) and HD-6, (GenBank Accession No.NP_(—)001917 herein incorporated by reference) are two human entericdefensins.

Defensins are toxic for a variety of infectious agents, such asGram-negative bacteria, Gram-positive bacteria, fungi, and certainenveloped viruses. Defensins act by forming pores in membranes of theinfectious agent and generating voltage-dependent channels.Antimicrobial activities of defensins include, but are not limited to,lysis of bacteria, fungi, or viruses; toxicity for bacteria, fungi orviruses; leukocyte (e.g., T cell) chemotaxis; and leukocyte (e.g.,neutrophil) recruitment. Without being bound by theory, defensins playan important role in the body's natural immunity against infections.Defensins are also cytotoxic for several normal and malignant cells. A“modified defensin” is a defensin that includes a modified arginineresidue. An “unmodified defensin” is a defensin that includes anyunmodified (native) arginine residue.

DNA: Deoxyribonucleic acid. DNA is a long chain polymer whichconstitutes the genetic material of most living organisms (some viruseshave genes composed of ribonucleic acid (RNA)). The repeating units inDNA polymers are four different nucleotides, each of which contains oneof the four bases, adenine, guanine, cytosine and thymine bound to adeoxyribose sugar to which a phosphate group is attached. Triplets ofnucleotides (referred to as codons) code for each amino acid in apolypeptide. The term codon is also used for the corresponding (andcomplementary) sequence of three nucleotides in the mRNA that istranscribed from the DNA.

Emphysema: A condition in which there is over-inflation of structures inthe lungs known as alveoli, or air sacs. This over-inflation resultsfrom a breakdown of the walls of the alveoli, which causes a decrease inrespiratory function and often, breathlessness. Early symptoms ofemphysema include shortness of breath and cough. Emphysema and chronicbronchitis together comprise chronic obstructive pulmonary disease(COPD).

Encode: A polynucleotide is said to “encode” a polypeptide if, in itsnative state or when manipulated by methods well known to those skilledin the art, it can be transcribed and/or translated to produce the mRNAfor and/or the polypeptide or a fragment thereof. The anti-sense strandis the complement of such a nucleic acid, and the encoding sequence canbe deduced therefrom.

Functionally Equivalent: Nucleic acid or polypeptide sequencealterations, for example in an ADP-ribosylated HNP-1 polypeptide, thatdo not alter a function of the nucleic acid or the polypeptide. In oneembodiment, the function is the promotion of neutrophil recruitment. Inanother embodiment the function is cytotoxicity. Such sequencealterations can include, but are not limited to, substitutions,deletions, base modifications, mutations, labeling, and insertions.

Immune cell: Any cell involved in a host defense mechanism. These caninclude, for example, T cells, B cells, natural killer cells,neutrophils, mast cells, macrophages, antigen-presenting cells,basophils, eosinophils, and neutrophils.

Immune response: A response of a cell of the immune system, such as aneutrophil, a B cell, or a T cell, to a stimulus. In one embodiment, theimmune response involves the phagocytosis of a microbe by a neutrophil,followed by the fusion of the neutrophil's azurophilic granules with thephagosome and/or the release of the contents of the neutrophil'sazurophilic granules extracellularly. In another embodiment, theresponse is specific for a particular antigen (an “antigen-specificresponse”).

Immune system deficiency: A disease or disorder in which the subject'simmune system is not functioning normally, quantitatively orqualitatively, or in which it would be useful to boost a subject'simmune response. In a non-limiting example, a subject with an immunesystem deficiency has a tumor (e.g., tumors of the brain, lung (e.g.,small cell and non-small cell), ovary, breast, prostate, colon, as wellas other carcinomas and sarcomas). In another non-limiting example, thesubject has an immune system deficiency, such as an immunodeficiencydisease resulting from a human immunodeficiency virus (HIV) infection.

Infectious agent: An agent that can infect a subject and/or cause aninfection, including, but not limited to, viruses, bacteria, and fungi.

Examples of infectious virus include: Retroviridae (for example, humanimmunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III,LAV or HTLV-III/LAV, or HIV-III; Paramyxoviridae (for example,parainfluenza viruses, mumps virus, measles virus, respiratory syncytialvirus); Orthomyxoviridae (for example, influenza viruses); andHerpesviridae (herpes simplex virus (HSV) 1 and HSV-2, varicella zostervirus, cytomegalovirus (CMV), herpes viruses).

Examples of infectious bacteria include: Helicobacter pyloris, Boreliaburgdorferi, Legionella pneumophilia, Mycobacteria sps (such as. M.tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae),Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis,Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus),Streptococcus agalactiae (Group B Streptococcus), Streptococcus(viridans group), Streptococcus faecalis, Streptococcus bovis,Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenicCampylobacter sp., Enterococcus sp., Haemophilus infiuenzae, Bacillusantracis, corynebacterium diphtheriae, corynebacterium sp.,Erysipelothrix rhusiopathiae, Clostridium perfringers, Clostridiumtetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturellamultocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillusmoniliformis, Treponema pallidium, Treponema pertenue, Leptospira, andActinomyces israelli.

Examples of infectious fungi include, but are not limited to,Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis,Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.

Other infectious organisms (such as protists) include: Plasmodiumfalciparum and Toxoplasma gondii.

Inflammation: A generalized response by the body as a result of tissuedamage. The tissue damage can be due to trauma, lack of blood supply,hemorrhage, autoimmunity, transplanted exogenous tissue, or infection.Inflammation includes the release of many components of the immunesystem (e.g., defensins, IL-1 and tumor necrosis factor), attraction ofcells to the site of the damage, swelling of tissue due to the releaseof fluid, and other processes.

During the inflammatory processes, a variety of soluble factors areinvolved in the recruitment of cells involved in the inflammatoryresponse through increased expression of cellular adhesion molecules andchemoattraction. Many of these soluble factors regulate the activationof both the resident cells (such as fibroblasts, endothelial cells,tissue macrophages, and mast cells) and the newly recruited inflammatorycells (such as monocytes, lymphocytes, neutrophils, and eosinophils). Inone embodiment, activated neutrophils release azurophilic granules thatcontain defensins. High defensin levels can be found in airwaysecretions of patients with inflammatory lung diseases.

Inflammatory Bowel Disease: Two separate diseases (Crohn's Disease andUlcerative Colitis) that cause inflammation of the bowel and can causearthritis or inflammation in joints. Crohn's Disease involvesinflammation of the colon or small intestines. Ulcerative Colitis ischaracterized by ulcers and inflammation of the lining of the colon. Theamount of the bowel disease usually influences the severity of arthritissymptoms.

Innate Immunity: Provides the first line of defense against many commonmicroorganisms and is essential for the control of common bacterialinfections. Includes antimicrobial peptides (e.g., defensins),epithelial barriers, phagocytic cells (neutrophils, macrophages),natural killer (NK) cells, the complement system, and cytokines thatregulate and coordinate many of the activities of these cells. Defensinsare present at the surface of epithelial cells, such as those lining thegut and the lungs, and in microbicidal organelles (e.g., azurophilicgranules) of the phagocytic cells of the hematopoietic system (e.g.,neutrophils and macrophages) and therefore are an important component tothe innate immune system. Innate immunity can be supplemented by theadministration of a modified defensin polypeptide. In one embodiment, anADP-ribosylated HNP-1 polypeptide, or otherwise modified defensin, isadministered to a subject. In another embodiment, a ribosyl-HNP-1polypeptide is administered to a subject.

Isolated: A biological component (such as a nucleic acid, peptide orprotein) that has been substantially separated, produced apart from, orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleicacids, peptides and proteins that have been “isolated” thus includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell, as well as chemically synthesizednucleic acids and proteins.

Leukocyte: Cells in the blood, also termed “white cells,” that areinvolved in defending the body against infective organisms and foreignsubstances. Leukocytes are produced in the bone marrow. There are fivemain types of white blood cells, subdivided between two main groups:polymorphonuclear leukocytes (neutrophils, eosinophils, basophils) andmononuclear leukocytes (monocytes and lymphocytes). When an infection ispresent, the production of leukocytes increases or leukocytes may berecruited to the site of infection.

Lymphocytes: A type of white blood cell that is involved in the immunedefense of the body. There are two main types of lymphocytes: B-cellsand T-cells.

Lysozyme: A highly basic 14.6 kDa protein (isoelectric point 11.1) of129 amino acids and containing 4 disulfide bonds. Lysozyme is aglycosidase that hydroyzes the bond between N-acteylmuramic acid andN-acetylglucosamine, thus cleaving an important polymer of the cell wallof many bacteria. Lysozyme is present in tears, saliva and in thelysosomes of phagocytic cells. It is an important antibacterial defense,particularly against Gram-positive bacteria.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Microorganism: An organism that can be seen only through a microscope.Microorganisms include bacteria, protozoa, algae, and fungi. Viruses arealso classified as microorganisms. A microorganism is also known as amicrobe.

Modified Arginine Residue: Any modification of an arginine in a protein.In one embodiment, the modification takes place on the guanidino groupof the arginine residue. Modification of the guanidino group includesbut is not limited to, the modification of an arginine residue byADP-ribosylation, acylation, alkylation, or polymer conjugation. Anarginine residue that is ADP ribosylated can be further modified forexample, by the pyrophosphatase/phosphatase cleavage of a pyrophosphateto yield a ribosyl-arginine residue. In one embodiment, a decarboxylatedarginine residue is a modified arginine residue known as agmatine(C₅H₁₄N₄).

Modulator: An agent that quantitatively or qualitatively modulates theactivity of a protein as measured by the change in an experimentalparameter. A modulator can be essentially any compound, such as apolypeptide, a hormone, a nucleic acid, a sugar, a lipid and the like.

Myeloperoxidase: A peroxidase found in the lysosomal granules ofleukocytes, particularly macrophages and neutrophils. Myeloperoxidase isan oxidoreductase that catalyzes the reaction of hydrogen peroxide andhalide ions to produce cytotoxic acids (such as hypochlorous acid) andother intermediates; these play a role in oxygen-dependent killing ofmicroorganisms and tumor cells.

Natural killer (NK) cell: These are large, usually granular, non-T,non-B lymphocytes, which kill certain tumor cells. NK cells areimportant in innate immunity to viruses and other intracellularpathogens, as well as in antibody-dependent cell-mediated cytotoxicity.

Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in eithersingle or double-stranded form, and unless otherwise limited,encompasses known analogues of natural nucleotides that hybridize tonucleic acids in a manner similar to naturally occurring nucleotides.

Neutrophil: Neutrophils are leukocytes of the PolymorphonuclearLeukocyte subgroup that are also known as granulocytes. Neutrophilscontain a lobed nucleus and abundant cytoplasmic granules that stainwith neutral dyes. Neutrophils form a primary defense against bacterialinfection. Like all the cells of the immune system, neutrophils areproduced in the bone marrow and circulate in the bloodstream. However,neutrophils move out of blood vessels into infected tissue in order toengulf and kill microorganisms (e.g., bacteria, fungus, virus).Neutrophils perform their function partially through the phagocytosis ofother cells and foreign substances. Neutrophils are recruited to a siteof infection by following a concentration gradient of chemoattractantsor cytokines.

Oligonucleotide: A linear polynucleotide sequence of up to about 200nucleotide bases in length, for example a polynucleotide (such as DNA orRNA) which is at least 6 nucleotides, for example at least 15, 50, 100or even 200 nucleotides long.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell. “Incubating” includes a sufficientamount of time for an agent to interact with a cell. “Contacting”includes incubating an agent in solid, or liquid, form with a cell.

A “therapeutically effective amount” is a quantity of a specificsubstance sufficient to achieve a desired effect in a subject beingtreated. For instance, this can be the amount necessary to inhibitviral, fungal, or bacterial replication or to measurably alter symptomsof the viral, fungal, or bacterial infection. When administered to asubject, a dosage will generally be used that will achieve target tissueconcentrations (for example, in lymphocytes) that has been shown toachieve a desired in vitro effect.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this disclosure are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15^(th) Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of modified antimicrobial peptides,such as alpha defensins or lysozyme.

In general, the nature of the carrier will depend on the particular modeof administration employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol, or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, pH buffering agents, or the like, for example sodiumacetate or sorbitan monolaurate.

Pneumonia: An inflammation of the lungs caused by a bacterial, viral, orfungal infection

Polynucleotide: A linear nucleotide sequence, including sequences ofgreater than 100 nucleotide bases in length.

Polypeptide: A polymer in which the monomers are amino acid residuesthat are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred in nature. The termpolypeptide or protein as used herein encompasses any amino acidsequence and includes, but may not be limited to, modified sequencessuch as ADP-ribosylated proteins, ribosyl-proteins, and glycoproteins.The term polypeptide is specifically intended to cover naturallyoccurring proteins, as well as those that are recombinantly orsynthetically produced.

Substantially purified polypeptide as used herein refers to apolypeptide that is substantially free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In one embodiment, the polypeptide is at least 50%, for example at least80%, free of other proteins, lipids, carbohydrates or other materialswith which it is naturally associated. In another embodiment, thepolypeptide is at least 90% free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In yet another embodiment, the polypeptide is at least 95% free of otherproteins, lipids, carbohydrates or other materials with which it isnaturally associated.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Any cDNA sequence variant will preferably introduce no more than twenty,and preferably fewer than ten amino acid substitutions into the encodedpolypeptide. Variant amino acid sequences may, for example, be 80%, 90%,95% or even 98% identical to the native amino acid sequence. Programsand algorithms for determining percentage identity can be found at theNCBI website.

Preventing or treating a disease: Preventing a disease refers toinhibiting completely, or in part, the development or progression of adisease, for example in a person who is known to have a predispositionto a disease. An example of a person with a known predisposition issomeone with a history of diabetes in the family, or who has beenexposed to factors that predispose the subject to a condition, such aslupus or rheumatoid arthritis. Treating a disease refers to atherapeutic intervention that ameliorates at least one sign or symptomof a disease or pathological condition, or interferes with apathophysiological process, after the disease or pathological conditionhas begun to develop.

Protein: A biological molecule encoded by a gene and comprised of aminoacids.

Pulmonary disease: A disease of the respiratory system, including thelungs and bronchial tree. Pulmonary diseases include cystic fibrosis,emphysema, asthma, sarcoidosis, chronic bronchitis, pulmonary fibrosis,pneumonia, and adult respiratory distress syndrome.

Pulmonary fibrosis: Chronic inflammation and progressive fibrosis ofalveolar walls, with steady, progressive shortness of breath, resultingin death from lack of oxygen or right heart failure.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein is more enriched thanthe peptide or protein is in its natural environment within a cell.Preferably, a preparation is purified such that the protein or peptiderepresents at least 50% of the total peptide or protein content of thepreparation.

Pyrophosphatase: An enzyme that catalyzes the hydrolysis ofpyrophosphate into two phosphate groups.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occur or was made artificially. Artificial combinationis often accomplished by chemical synthesis or, more commonly, by theartificial manipulation of isolated segments of nucleic acids, e.g., bygenetic engineering techniques. Similarly, a recombinant protein is oneencoded by a recombinant nucleic acid molecule.

Sarcoidosis: A disease of unknown cause in which inflammation,consisting of granulomas, occurs in lymph nodes, lungs, liver, eyes,skin, or other tissues. Tissue samples from involved organs showgranulomas, which are clusters of macrophages, lymphocytes, andmultinucleated giant cells. Possible causes of sarcoidosis include ahypersensitivity response, a genetic predisposition, infection, orchemicals.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes both human and non-human mammals.

T Cell: A white blood cell critical to the immune response. T cellsinclude, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ Tlymphocyte is an immune cell that expresses a marker on its surfaceknown as “cluster of differentiation 4” (CD4). These cells, also knownas helper T cells, help orchestrate the immune response, includingantibody responses and killer T cell responses. CD8 T cells express the“cluster of differentiation 8” (CD8) marker. In one embodiment, a CD8 Tcell is a cytotoxic T lymphocyte. In another embodiment, a CD8 T cell isa suppressor T cell.

T cell chemotaxis: The directed locomotion of a T cell along aconcentration gradient of chemotactically active factors, such ascytokines. Cells showing positive chemotaxis move towards areas withhigher concentrations of these agents, those showing negative chemotaxismove away from these areas.

An increase in T cell chemotaxis includes, but may not be limited to, anincrease in the distance or rate of T cell migration, an increase in thenumber of T cells migrating, an increase in the types of T cellsmigrating in a sample in response to a chemotactic stimulus, as comparedto a control sample which does not receive the chemotactic stimulus.

Therapeutically effective dose: A dose sufficient to preventadvancement, or to cause regression of the disease, or which is capableof relieving symptoms caused by the disease, such as pain or swelling.

Treatment: Refers to both prophylactic inhibition of initial infectionor disease, and therapeutic interventions to alter the natural course ofan untreated infection or disease process, such as a tumor growth or aninfection with a bacteria.

Ulcerative colitis: An Inflammatory Bowel Disease characterized byulcers and inflammation of the lining of the colon.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

Modified Immunomodulatory Polypeptides

A composition is provided herein that is a polypeptide that modulates animmune response and that includes a modified arginine residue. In oneembodiment, the polypeptide that modifies an immune response is anantimicrobial agent. A specific, non-limiting example of anantimicrobial agent is a defensin polypeptide, for example an alphadefensin.

In one specific embodiment, the alpha defensin is a vertebratepolypeptide. In another specific embodiment, the alpha defensinpolypeptide is a mammalian polypeptide. In yet another specificembodiment, the alpha defensin polypeptide is from a human. In otherembodiments, the alpha defensin polypeptide is from a monkey, a rabbit,a rat, a cat, a dog, a pig, a sheep, or a mouse. In a specific,non-limiting example, the alpha defensin is human neutrophil peptide(HNP)-1. In other specific, non-limiting examples, the alpha defensinpolypeptide is HNP-2, HNP-3, HNP-4, HD-5, HD-6, or Def-X.

The alpha defensins include HNP-1, HNP-2, HNP-3, HNP-4, HD-5, HD-6, andDef-X. HNP-1, HNP-2, and HNP-3 are products of the same 94 amino acidprepro-protein. In one embodiment, this protein has the followingsequence:

(SEQ ID NO: 1, see also GenBank Accession No.P11479, herein incorporated by reference)MRTLAILAAILLVALQAQAEPLQARADEVAAAPEQIAADIPEVVVSLAWDESLAPKHPGSRKNMDCYCRIPACIAGERRYGTCIYQGRLWAFCC;.

HNP-1 is one member of the family of alpha defensins produced bycleavage of the preproprotein. In one embodiment, HNP-1 has a sequenceas set forth as:

ACYCRIPACIAGERRYGTCIYQGRLWAFCC;. (SEQ ID NO: 2)

HNP-2 is another member of the family of alpha defensins produced bycleavage of the preproprotein. In one embodiment, HNP-2 has a sequenceas set forth as:

CYCRIPACIAGERRYGTCIYQGRLWAFCC;. (SEQ ID NO: 3)

HNP-3 is a third member of the family of alpha defensins produced bycleavage of the preproprotein. In one embodiment, HNP-3 has a sequenceas set forth as:

DCYCRIPACIAGERRYGTCIYQGRLWAFCC;. (SEQ ID NO: 4)

HNP-4 is an alpha defensin that is the product of a prepro-proteinhaving a sequence as set forth as:

(SEQ ID NO: 5, see also GenBank Accession No.NP_001916, herein incorporated by reference)MRIIALLAAILLVALQVRAGPLQARGDEAGQEQRGPEDQDISISFAWDKSSALQVSGSTRGMVCSCRLVFCRRTELRVGNCLIGGVSFTYCCTRVD.

In one embodiment, HNP-4 has a sequence as set forth as:

VCSCRLVFCRRTELRVGNCLIGGVSFTYCCTRVD;. (SEQ ID NO: 6)

HD-5 is produced by cleavage of the following prepro-protein having asequence as set forth as:

(SEQ ID NO: 7, GenBank Accession No.NP_066290, herein incorporated by reference)MRTIAILAAILLVALQAQAESLQERADEATTQKQSGEDNQDLAISFAGNGLSALRTSGSQARATCYCRTGRCATRESLSGVCEISGRLYRLCCR;.

In one embodiment, HD-5 has a sequence as set forth as:

ATCYCRTG RCATRESLSG VCEISGRLYR LCCR;. (SEQ ID NO: 8)

HD-6 is produced by cleavage of the following prepro-protein having asequence as set forth as:

(SEQ ID NO: 9, GenBank Accession No.NP_001917, herein incorporated by reference)MRTLTILTAVLLVALQAKAEPLQAEDDPLQAKAYEADAQEQRGANDQDFAVSFAEDASSSLRALGSTRAFTCHCRRSCYSTEYSYGTCTVMGINHRFC CL;.

In one embodiment, HD-6 has a sequence as set forth as:

TCHCRRSCYS TEYSYGTCTV MGINHRFCCL;. (SEQ ID NO: 10)

Def-X is produced by cleavage of the following prepro-protein having asequence as set forth as:

(SEQ ID NO: 13, see U.S. Pat. No. 6,329,340,herein incorporated by reference)MRTLTLLSAFLLVALQAWAEPLQARAHEMPAQKQPPADDQDVVIYFSGDDSCSLQVPGSTKGLICHCRVLYCIFGEHLGGTCFILGERYPICCY.

In one embodiment, Def-X has a sequence as set forth as:

ICHCRVLYCIFGEHLGGTCFILGERYPICCY (SEQ ID NO: 14)

As disclosed herein, a modified alpha defensin includes an arginineresidue modified at the guanidino group. In one embodiment, the arginineresidue is modified by ADP-ribosylation to form an ADP-ribosylatedarginine residue. An alpha defensin including at least oneADP-ribosylated residue is referred to as an ADP-ribosylated defensin.In several embodiments, the ADP-ribosylated alpha defensin includes atleast one, at least two, at least three, or at least fourADP-ribosylated arginine residues.

An ADP-ribose is covalently attached to another compound byADP-ribosylation. Two mammalian ARTs, ART-1 and ART-5, are known tospecifically modify arginine residues in proteins. In one embodiment,the ART substrate (the source of the ADP-ribose) is NAD.

ADP-ribose acceptors of the five known mammalian ARTs include basic andarginine-rich proteins that are involved in host defense (e.g.,lymphocyte activation, neutrophil chemotaxis). ADP-ribose acceptors thatcontain an arginine can also be modified by an ART, but need not bearginine rich or basic. ADP-ribose acceptors include, but may not belimited to, alpha defensins (HNP-1, HNP-2, HNP-3, HNP-4, HD-5, HD-6,Def-X), beta defensins (hBD1, hBD-2, hBD-3, hBD-4), Major Basic Protein,Eosinophil Cationic Protein, Human Cathelicin LL-37 (hCAP18), andlysozyme. Thus, an ADP-ribosyltransferase can be used to produceADP-ribosylated immunomodulatory polypeptides, such as a defensin.

In one specific, non-limiting example, the alpha defensin is HNP-1 andincludes at least one ADP-ribosylated arginine residue. In anotherspecific, non-limiting example, the alpha defensin is HNP-1 that isADP-ribosylated on an arginine residue at position 14 of SEQ ID NO:2. Inanother embodiment, HNP-1 is ADP-ribosylated on an additional arginineresidue and the ADP-HNP-1 includes at least one, at least two, at leastthree, or at least four ADP-ribosylated arginine residues.

In another embodiment, a pyrophosphate is cleaved in an ADP-ribosylatedarginine, followed by the removal of phosphate, to yield aribosyl-arginine. An alpha defensin including at least oneribosyl-arginine is referred to as a ribosylated alpha defensin. In oneembodiment, a pyrophosphate linkage is cleaved by apyrophosphatase/phosphatase and phosphate is removed from anADP-ribosylated alpha defensin polypeptide. In another embodiment, theribosylated alpha defensin is HNP-1. In other embodiments, theribosylated alpha defensin is ribosylated-HNP-2, -HNP-3, -HNP-4, -HD-5,-HD-6, or -Def-X to yield ribosyl-HNP-2, -HNP-3, -HNP-4, -HD-5, -HD-6,-Def-X.

The modification of an arginine residue on its guanidino group alsoincludes, but is not limited to, acylation, alkylation, or polymerconjugation. In one embodiment, a decarboxylated arginine residue is amodified arginine residue known as agmatine (C₅H₁₄N₄).

The defensin polypeptide can be modified by the addition of arginineresidues to the polypeptide. In one embodiment, the at least onearginine is added to the amino terminal end of the polypeptide. In onespecific, non-limiting example two arginines are added to the aminoterminal end of the alpha defensin polypeptide. In another embodiment,at least one arginine is added to the carboxy terminal end of thepolypeptide. In one specific, non-limiting example, two arginines areadded to the carboxy terminal end of the polypeptide. In yet anotherembodiment, at least one arginine is added to both the amino and carboxyterminal ends of the polypeptide.

Antimicrobial agents, other than alpha defensins, are also encompassedby the disclosure. Examples of antimicrobial agents, other than alphadefensins, that are modified by ADP-ribosylation or ribosylationinclude, but are not limited to, lysozyme, BPI, beta defensins (hBD1,hBD-2, hBD-3, hBD-4), Major Basic Protein, Eosinophil Cationic Protein,Human Cathelicin LL-37 (hCAP18), and myeloperoxidase.

Pharmaceutical Compositions and Administration

Pharmaceutical compositions that include an ADP-ribosylatedantimicrobial agent or a ribosyl-antimicrobial agent can be formulatedwith an appropriate solid or liquid carrier, depending upon theparticular mode of administration chosen. A specific, non-limitingexample of an ADP-ribosylated antimicrobial agent includes anADP-ribosylated defensin polypeptide, for example an ADP-ribosylatedalpha defensin. A specific, non-limiting example of a ribosylatedantimicrobial agent includes a ribosylated defensin polypeptide, forexample a ribosylated alpha defensin. The pharmaceutically acceptablecarriers and excipients useful in this disclosure are conventional. Forinstance, parenteral formulations usually comprise injectable fluidsthat are pharmaceutically and physiologically acceptable fluid vehiclessuch as water, physiological saline, other balanced salt solutions,aqueous dextrose, glycerol or the like. Excipients that can be includedare, for instance, other proteins, such as human serum albumin or plasmapreparations. If desired, the pharmaceutical composition to beadministered can also contain minor amounts of non-toxic auxiliarysubstances, such as wetting or emulsifying agents, preservatives, and pHbuffering agents and the like, for example sodium acetate or sorbitanmonolaurate.

Medicinal and pharmaceutical agents, for instance immunostimulants, alsocan be included. Immunostimulants include, but are not limited to,cytokines, such as Macrophage Inflammatory Protein (MIP)-β, IL-1, IL-8,IL-10, granulocyte-macrophage colony stimulating factor, granulocytecolony stimulating factor, neurokinin, and tumor necrosis factor-alpha,for example.

The dosage form of the pharmaceutical composition will be determined bythe mode of administration chosen. For instance, in addition toinjectable fluids, topical, inhalation, oral and suppositoryformulations can be employed. Topical preparations can include eyedrops, ointments, sprays, patches and the like. Inhalation preparationscan be liquid (e.g., solutions or suspensions) and include mists, spraysand the like. Oral formulations can be liquid (e.g., syrups, solutionsor suspensions), or solid (e.g., powders, pills, tablets, or capsules).Suppository preparations can also be solid, gel, or in a suspensionform. For solid compositions, conventional non-toxic solid carriers caninclude pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in the art.

The pharmaceutical compositions that include an ADP-ribosylated defensinpolypeptide, or ribosyl defensin molecule, can be formulated in unitdosage form, suitable for individual administration of precise dosages.In one specific, non-limiting example, a unit dosage can contain fromabout 1 ng to about 1 mg of ADP-ribosylated HNP-1 polypeptide. Theamount of active compound(s) administered will be dependent on thesubject being treated, the severity of the affliction, and the manner ofadministration, and is best left to the judgment of the prescribingclinician. Within these bounds, the formulation to be administered willcontain a quantity of the active component(s) in amounts effective toachieve the desired effect in the subject being treated.

The compounds of this disclosure can be administered to humans or otheranimals on whose tissues they are effective in various manners such astopically, orally, intravenously, intramuscularly, intraperitoneally,intranasally, intradermally, intrathecally, subcutaneously, viainhalation or via suppository. The particular mode of administration andthe dosage regimen will be selected by the attending clinician, takinginto account the particulars of the case (e.g. the subject, the disease,the disease state involved, and whether the treatment is prophylactic).Treatment can involve daily or multi-daily doses of compound(s) over aperiod of a few days to months, or even years.

A therapeutically effective amount of a modified defensin polypeptide,such as an ADP-ribosylated alpha defensin polypeptide or a ribosyl-alphadefensin polypeptide, can be the amount of ADP-ribosylated alphadefensin polypeptide or ribosyl-alpha defensin polypeptide necessary tomodulate the immune system of a subject. Specific immunostimulatoryeffects that can be caused by ADP-ribosylated alpha defensin polypeptideor ribosyl-alpha defensin polypeptide are described herein. In someembodiments, an immunomodulatory amount of an ADP-ribosylated defensinpolypeptide or ribosyl-defensin polypeptide is an amount sufficient tostimulate an immune response for instance, increased T cell chemotaxisor promotion of neutrophil recruitment.

A therapeutically effective amount of modified defensin polypeptide,such as an ADP-ribosylated defensin polypeptide or ribosyl-defensinpolypeptide, can be administered in a single dose, or in several doses,for example daily, during a course of treatment. However, the effectiveamount of ADP-ribosylated defensin polypeptide or ribosyl-defensinpolypeptide will be dependent on the subject being treated, the severityand type of the affliction, and the manner of administration of thetherapeutic(s).

Site-specific administration of the disclosed compounds can be used, forinstance by applying the modified defensin polypeptide (for example aribosyl-alpha defensin polypeptide) to a region of inflammation, aregion of infection, or a region suspected of being prone toinflammation or infection.

Also encompassed by the present disclosure are pharmaceuticalcompositions that include ADP-ribosylated or ribosylated antimicrobialagents, other than alpha defensins, and a pharmaceutically acceptablecarrier. Such pharmaceutical compositions include ADP-ribosylated orribosylated antimicrobial agents such as lysozyme, BPI, beta defensins(hBD1, hBD-2, hBD-3, hBD-4), Major Basic Protein, Eosinophil CationicProtein, Human Cathelicin LL-37 (hCAP18), and myeloperoxidase.

The present disclosure also includes combinations of a modifiedantimicrobial agent, such as an ADP-ribosylated alpha defensinpolypeptide or ribosyl-alpha defensin polypeptide, with one or moreother agents useful in the treatment of an immune-related disorder,condition, or disease. For example, the compounds of this disclosure canbe administered in combination with effective doses of modifiedantimicrobial agents other than defensins, immunostimulants, anti-tumoragents, anti-inflammatory agents, anti-infectives, and/or vaccines. Theterm “administration in combination” or “co-administration” refers toboth concurrent and sequential administration of the active agents. Asubject that is infected with an infectious agent, or displays an immunesuppression, will be a candidate for treatment using the therapeuticmethods disclosed herein, as described below.

Method of Modulating an Immune Response

A method is disclosed herein of modulating an immune response of anantimicrobial agent such as, for example, a defensin polypeptide or alysozyme. Defensin polypeptides are antimicrobial peptides that areinvolved in the innate immune defense and are cytotoxic for microbessuch as bacteria, fungi, and certain types of viruses. In addition, theystimulate IL-8 release from neighboring cells and induce an increase inT cell chemotaxis. As disclosed herein, the modification of an arginineresidue in a defensin polypeptide can alter its antimicrobial activityand/or modify an immune response. Thus, a method is provided herein formodulating the anti-microbial activity of a defensin, such as an alphadefensin. In several embodiments, ADP-ribosylating a defensin orribosylating a defensin increases the anti-microbial activity of thedefensin. In several specific, non-limiting examples, the alphadefensin, includes but is not limited to, HNP-1, HNP-2, HNP-3, HNP-4. Inspecific, non-limiting examples, the modified alpha defensin isADP-ribosylated HNP-1 or ribosylated HNP-1.

The antimicrobial activity can be antibacterial, antifungal, orantiviral activity. In one embodiment, an alteration in antimicrobialactivity is a decrease in antimicrobial activity. In severalnon-limiting examples, the change in antimicrobial activity is at leastabout a 50% decrease, at least about a 75% decrease, at least about an80% decrease, at least about a 90% decrease, at least about a 95%decrease, at least about a 98% decrease, or at least about a 100%decrease. In one embodiment, an alteration in antimicrobial activity isan increase in antimicrobial activity. In several specific, non-limitingexamples, the change in antimicrobial activity is at least about a 50%increase, at least about a 75% increase, at least about an 80% increase,at least about a 90% increase, at least about a 95% increase, at leastabout a 98% increase, or at least about a 100% increase. In oneembodiment, the altered antimicrobial activity is increased lysis ofbacteria, fungi, or viruses.

In another embodiment, the altered antimicrobial activity is an increasein cytokine production. The increase in cytokine expression can be anincrease in cytokine secretion, expression, and/or release. In onespecific, non-limiting example, the cytokine is IL-8.

In yet another embodiment, the altered antimicrobial activity is anincrease in the recruitment of inflammatory cells. In one specific,non-limiting example, the inflammatory cells are neutrophils. In afurther embodiment, the altered antimicrobial activity is an increase ininflammatory cell chemotaxis. In one specific, non-limiting example theinflammatory cells are T cells.

In one embodiment, the antimicrobial activity is increased as comparedto unmodified (native) alpha defensin polypeptide. In a furtherembodiment, the antimicrobial activity is decreased as compared tounmodified (native) alpha defensin polypeptide. In one specific,non-limiting example, the modified alpha defensin is an ADP-ribosylatedHNP-1 polypeptide and the unmodified alpha defensin isnon-ADP-ribosylated HNP-1 polypeptide. In another specific, non-limitingexample, the modified alpha defensin is a ribosyl-HNP-1 polypeptide andthe unmodified alpha defensin is non-ribosyl-HNP-1 polypeptide.

In one embodiment, a method of modifying an immune response against amicrobe is provided. The method includes administering a therapeuticallyeffective amount of a modified defensin to a subject infected with or atrisk of being infected with the microbe, thereby modulating the immuneresponse against the microbe. In one embodiment, modifying immuneresponse includes decreasing cytotoxic activity. In another embodiment,modification of the immune response includes increasing neutrophilrecruitment.

Neutrophil recruitment can be measured by any method known to one ofskill in the art. In one embodiment, promotion of neutrophil recruitmentis measured by the release of IL-8 from cells. In one specific,non-limiting example, IL-8 release is measured by indirect ELISA(enzyme-linked immunosorbent assay).

In a further embodiment, modification of an immune response includesincreasing lymphocyte chemotaxis. Thus, the administration of a modifieddefensin, such as a modified alpha defensin, modulates T cellchemotaxis. T cell chemotaxis can be measured by any means known to oneof skill in the art, but is generally measured by measuring the lengthof migration of the T cells, the number of migrating T cells, or both.In one specific, non-limiting example, T cell migration is measured invitro, such as by measuring T cell migration from one cell culturechamber to another cell culture chamber through a porous membrane.

In another embodiment, modification of the immune response includesaltering an inflammatory response. An inflammatory response can bemeasured by any means known to one of skill in the art. In oneembodiment, an inflammatory response is measured by assessing the numberof activated T cells present in the sample. In another embodiment, aninflammatory response is measured by a change in cytokine production,such as a change in IL-8 production. In several embodiments, increasedcytokine production is a 100%, 200%, or 300% increase in cytokineproduction in the presence of a modified defensin.

The subject can be any mammal. In one embodiment, the subject is ahuman. In other embodiments, the subject may be a monkey, a rabbit, arat, a pig, a sheep, a dog, a cat, or a mouse. In one embodiment, thesubject has a disease, such as a pulmonary disease. Specific,non-limiting examples of pulmonary diseases are emphysema, adultrespiratory distress syndrome, asthma, bronchopulmonary dysplasia,chronic bronchitis, sarcoidosis, pulmonary fibrosis, or cystic fibrosis.In another embodiment, the subject is infected with a pathogen, such asa bacteria, fungus, or virus. Specific, non-limiting examples ofbacterial infections affecting the lungs are pneumonia, or tuberculosis.

In another embodiment, the subject has a tumor, such as a benign or amalignant tumor. Specific, non-limiting examples are lung, intestine,colon, breast, ovarian, uterine, prostate, testicular, or liver tumors.

In another embodiment, the subject has an intestinal disease. Specific,non-limiting examples of intestinal diseases are Inflammatory BowelDiseases such as Crohn's Disease and Ulcerative Colitis.

In yet another embodiment, the subject is immunodeficient. In onespecific, non-limiting example, the subject is infected with animmunodeficiency virus, such as a human immunodeficiency virus (e.g.,HIV-1 or HIV-2). In a further embodiment, the subject has an autoimmunedisorder.

Additional Methods

A method is disclosed herein for inhibiting the cytotoxic activity of anon-ADP-ribosylated alpha defensin polypeptide in a subject. The methodincludes administering to a subject a therapeutically effective amountof an ADP-ribosylated defensin polypeptide or a ribosylated defensinpolypeptide to inhibit the cytotoxic activity of a non-ADP-ribosylatedalpha defensin polypeptide. In one embodiment, the ADP-ribosylateddefensin polypeptide or ribosylated defensin polypeptide is an alphadefensin polypeptide. In another specific, non-limiting example, thealpha defensin polypeptide is HNP-1. In a specific embodiment, anarginine residue at position 14 of the HNP-1 sequence as in SEQ ID NO:2is ADP-ribosylated.

In one embodiment, the cytotoxic activity is measured by the ability ofan alpha defensin polypeptide to lyse a cell. In several embodiments,the lysed cell is a normal cell, a malignant cell, or a cell that isresistant to host defense mechanisms.

In one embodiment, cell lysis is measured by the number of viable cellsremaining in a sample containing ADP-ribosylated alpha defensinfollowing an incubation period, compared to a control sample with thesame starting number of cells but that contains non-ADP-ribosylatedalpha defensin.

A method is provided herein for screening for an agent that affectsADP-ribosylation of a defensin polypeptide. The method includes exposureof a cell that expresses an adenosine-diphosphate ribosyltransferasewith the agent in the presence of the defensin, and evaluating theADP-ribosylation or ribosylation of the alpha defensin (Zolkiewska andMoss J Biol Chem; 34: 25273-25276, 1993).

In one embodiment, ADP-ribosylation or ribosylation is increased ascompared to a control. In another embodiment, ADP-ribosylation orribosylation is decreased as compared to a control. Suitable controlsinclude, but are not limited to, a standard curve, a known amount ofADP-ribosylated defensin polypeptide, or a known amount of ribosylateddefensin polypeptide. A suitable control also includes, but is notlimited to, a cell not contacted with the agent, or a cell that does notexpress the adenosine-diphosphate ribosyltransferase.

To assess the ability of an agent to inhibit or alter ADP-ribosylationor ribosylation of a defensin, a series of assays can be carried out inthe presence of varying concentrations of the putative modulatorycompound (including zero concentration), and the extent ofADP-ribosylation or ribosylation of the defensin is determined for eachassay.

Suitable agents for screening include, but are not limited to smallmolecules, peptides, antibodies, chemical compounds, antisense nucleicacids, or peptidomimetics. The test compound may also be a combinatoriallibrary for screening multiple compounds. In addition, compoundsidentified in the method of the invention can be further evaluated,detected, cloned, sequenced, and the like, either in solution or afterbinding to a solid support, by any method usually applied to thedetection of a specific DNA sequence, such as PCR, oligomer restriction(Saiki et al., Bio/Technology 3:1008-1012, 1985), allele-specificoligonucleotide (ASO) probe analysis (Conner et al., Proc. Natl. Acad.Sci. USA 80:278, 1983), oligonucleotide ligation assays (OLAs)(Landegren et al., Science 241:1077, 1988), and the like. Moleculartechniques for DNA analysis have been reviewed (Landegren et al.,Science 242:229-237, 1988).

The invention is illustrated by the following non-limiting Examples.

EXAMPLES Example 1 Cloning, Expression and Purification of RecombinantART-1 with a FLAG Tag

Wild-type adenosine-diphosphate ribosyltransferase (ART)-1 was clonedfrom a rabbit muscle cDNA library by polymerase chain reaction usingforward (5′-ACGTACAAGCTTAGCCACCTG GTGACACGTCGAGAC, SEQ ID NO:11) andreverse (5′-ACGTACGGTACCGTCCAGGTGGC AGGGCCTAGACTT, SEQ ID NO:12)primers. PCR products were digested with Hind III and Kpn I (RocheMolecular Biochemicals, Indianapolis, Ind.), and then subcloned into apFLAG-MAC (Sigma, St. Louis, Mo.) expression vector that was used totransform E. coli ampicillin resistant BL-21 (DE 3) competent cells(Novagen Inc, Madison, Wis.). Single colonies were grown in LuriaBertani (LB) medium with ampicillin (50 μg/ml) to a change in opticaldensity of 0.4, at 600 nm, before adding isopropyl-D thiogalactoside(IPTG) (final concentration, 0.5 mM). Following incubation for 2 hoursat 30° C., bacteria were pelleted by centrifugation (15 minutes at3,200×g, 4° C.), resuspended in 50 M Tris (pH 8.0), 5 mM EDTA, lysozyme(0.25 mg/ml), 1.5 M NaCl, 0.1 M MgCl₂, DNase I (0.02 μg/ml) and proteaseinhibitor cocktail (Boheringer Mannheim, Mannheim Germany) (50 μg/ml),sonicated and centrifuged (45 minutes at 12,500×g, 4° C.). Supernatantswere applied to anti-FLAG M2 affinity gel (Sigma) and eluted followingthe manufacturer's instructions. Eluates were concentrated bycentrifugation at 4300×g (4° C.) using Centricon 10 columns (Millipore,Bedford, Mass.).

Example 2 ADP-Ribosyltransferase Assay

The ADP-ribosyltransferase activity of ART-1 was assayed as described(Weng et al., J. Biol. Chem. 274:31797-31803, 1999; Okazaki et al.,Blood. 88:915-21, 1996). The reaction was carried out in 0.3 ml of 50mmol/l potassium phosphate, pH 7.5, with 20 mmol/l agmatine and 0.1mmol/l NAD, [adenine-U-¹⁴C]NAD (281 mCi/mmol). After incubation at 30°C., samples (0.1 ml) were applied to a 1 ml column of Dowex AG 1-X2(Biorad, Hercules, Calif.). [¹⁴C]ADP-ribosylagmatine was eluted with 5ml of water for radioassay.

For HNP modification by ART-1, 2 μg of synthetic HNP-1 (Bachem,Torrance, Calif.) was incubated in 200 μl of 50 mM potassium phosphate,pH 7.5, with ART-1 (24 pmol/minute/mg) and 3 mM NAD⁺. After overnightincubation at 30° C., the mixture was solubilized in 6 M guanidine andapplied to a Vydac C18 column (Nest Group, Southboro, Mass.)equilibrated with solution A (HPLC grade water, 0.1% trifluoroaceticacid). ADP-ribose-HNP-1 was separated from the unmodified form bygradient elution with solution B (100% isopropanol, 0.2% trifluoroaceticacid) at a flow rate of 1 ml/minute: 100% solution A, 0-20 minutes,0-60% linear gradient of solution B from 20 to 80 minutes, and 95%solution B from 60 to 85 minutes (FIG. 1). Fractions were analyzed byMALDI mass spectrometry as described (Rivera-Nieves et al., J Biol.Chem. 274:19525-19531, 1999).

Peptides with a mass of 3,441 daltons (retention time 48.5 minutes),consistent with HNP-1 (calculated mass equivalent to 3,442 daltons), andpeptides with a mass of 3,983 daltons (retention time 46.5 minutes),consistent with mono-ADP-ribosyl-HNP (calculated mass equivalent to3,983 daltons), were identified. Modified HNP exhibited an absorbancepeak at 258 nm, which was not exhibited by HNP (FIG. 1D, inset). TheADP-ribose was released from modified HNP by incubating the HNP withADP-ribosylarginine hydrolase, yielding the unmodified defensin.

For ADP-ribosylation of intact cells, intact C2C12 or G8 cells attachedto culture plates were incubated for 1 hour at 37° C. in PBS (2 ml for a100 mm plate, 1 ml for a 60 mm plate), 5 mM [adenylate—³²P]NAD (4Ci/mmol) and 1 mM ADP-ribose, washed twice with PBS, lysed with 0.5 mlof lysis buffer (3% SDS, 0.1 M sodium acetate (pH 6.8), 5 mM EDTA),scraped and boiled for 10 minutes. Protein concentration was determinedfor the cells harvested from each plate. For SDS-PAGE analysis, 100 μgof protein was precipitated with 10% trichloroacetic acid, suspended ingel-loading buffer and subjected to SDS-PAGE on 8% gel. Gels werestained for protein, dried and exposed to Kodak-X-Omat film for 24 hoursat −80° C. with an intensifying screen. The time-course of labeling of a97 kilodalton protein was measured using a ProsphorImager (MolecularDynamics) (Zolkiewska and Moss, J. Biol. Chem. 34: 25273-25276, 1993).

Example 3 Electrospray Mass Spectrometric Analysis of ADP-RibosylatedHNP-1

To identify the modified amino acid in HNP-1, reduced ADP-ribosylatedHNP-1 was digested with trypsin and the fragments were analyzed by massspectrometry. Samples (2 μg) of ADP-ribosyl-HNP-1 and HNP-1 were reducedby incubation in 50 μl of 100 mM Tris-HCl (pH 8), 1 mM EDTA, and 20 mMdithiothrietol for 1 hour at 37° C. before incubation for 6 hours at 37°C. with trypsin (1 μg) (Sigma) in 100 mM, NH₄HCO₃ (total volume, 100μl). The modified site was identified by electrospray mass spectrometryas described (Rivera-Nieves et al., J Biol. Chem. 274:19525-19531,1999).

A peak representing a peptide with a mass of 1,626.3 daltons, compatiblewith ADP-ribosylated fragment P₆₋₁₅ (calculated mass equivalent to1,626.3 daltons), was observed. This peak, unique to ADP-ribosylatedHNP-1, was generated by the inhibition of trypsin cleavage of thedefensin peptide as a result of ADP-ribosylation at arginine 14.Spectral analysis was consistent with the presence of adenosine in thepeptide.

Example 4 Cytotoxicity Assay

The antibacterial activity of various concentrations of HNP-1 andADP-ribosyl-HNP-1 (16 nM, 32 nM, 64 nM, 128 nM, 256 nM) on Escherichiacoli ATCC43827 (American Type Culture Collection, Rockville, Md.) wasevaluated by the radial diffusion assay (Takemura et al., Antimicrob.Agents Chemother. 40: 2280-2284, 1996). Antimicrobial activity waspositively correlated with the diameter of a zone cleared of E. coligrowth (minus the diameter of the central well (3 mm)). The results(FIG. 2A) indicate that ADP-ribosylated HNP-1 had a reducedantibacterial activity, compared to unmodified HNP-1, for E. coliATCC43827, as measured by the radial diffusion assay.

Example 5 Chromium Release Assay

Chromium (⁵¹Cr)-labeled A549 cells (American Type Culture Collection)were incubated (18 hours, 37° C.) in 100 μl of serum-free RPMI (GibcoFluids Inc., Rockville, Md.) containing various concentrations of HNP-1,synthetic HNP-1, or ADP-ribosyl-HNP-1 (1.5 μM, 3 μM, 6 μM, 12 μM, 24 μM)to quantify defensin cytotoxicity. Cytotoxicity was positivelycorrelated with the amount of chromium released from lysed cells(Panyutich et al., Am. J. Respir. Cell. Mol. Biol. 12:351-357, 1995).Percent of lysis was calculated as(cpm_(exp)−cpm_(spont))/(cpm_(max)−cpm_(spont))×100 (exp, experimentalconditions; max, maximal release; spont, spontaneous release). Theresults (FIG. 2B) indicate that ADP-ribosylated HNP-1 was less cytotoxicthan unmodified HNP-1 for A549 cells.

Example 6 Radial Diffusion and Chromium Release Assays to Measure theEffect of ADP-Ribosylated

HNP-1 on HNP-1 Cytotoxic Activity HNP-1 (100 nM) was incubated for 1hour at 37° C. with various concentrations of ADP-ribosylated HNP-1(0-800 nM) before the initiation of the E. coli radial diffusion assay.The radial diffusion assay was performed as described in Example 4.

HNP-1 (12 μM) was incubated with various concentrations ofADP-ribosyl-HNP-1 (0-12 μM) for 1 hour at 37° C. before the initiationof the chromium release assay. The chromium release assay was performedas described in Example 5. HNP-1 and ADP-ribosyl-HNP-1 (0-12 μM) wereincluded as controls.

The radial diffusion and chromium release assays demonstrated thatADP-ribosylated HNP-1 blocked the cytotoxic activity of HNP-1 in aconcentration-dependent manner (FIG. 3). Another experiment, using ahigher HNP-1 concentrations (24 μM), showed similar results.

Example 7 IL-8 Production by A549 Cells

A549 cells (3×10⁴ cells per well) were incubated in a 96-well plate in200 μl of serum-free RPMI medium (Gibco Fluids Inc) containingADP-ribosyl-HNP-1 or HNP-1 (0.25, 0.75, 1.5, 3 μM). Culture medium wassampled after 12 or 24 hours of incubation and IL-8 content in themedium was assayed by indirect ELISA (FIGS. 4A and 4B) according to themanufacturer's instructions (R &D System Inc. Minneapolis, Minn.). Atconcentrations of 0.75 and 1.5 μM, IL-8 release into the medium wassignificantly higher with ADP-ribosylated HNP-1 than with the unmodifiedpeptide (P=0.01). At a higher peptide concentration, 3 μM, nodifferences were observed.

Example 8 Chemotaxis Assay

CD3⁺ T-cells were isolated from human peripheral blood prepared byleukapheresis (NIH, Department of Transfusion Medicine, Bethesda, Md.)(Chertov et al, J. Biol. Chem. 271:2935-2940, 1996) and suspended inmigration medium (RPMI 1640, 0.5% bovine serum albumin, 25 mM HEPES).Inserts coated with collagen IV (Becton Dickinson Labware, Bedford,Mass.) were placed into 24-well culture plates to form upper and a lowerchambers in each well. Upper chambers were wetted with migration medium,then 500 μl of migration medium with or without ADP-ribosyl-HNP-1 orHNP-1 (0.025 to 25 nM) were added to the lower chamber. Cells were addedto the upper chamber and plates were incubated at 37° C. in 5% CO₂ for 4hours. Cells incubated in the presence or absence of (macrophageinflammatory protein-1β) MIP-1β (5 ng/ml) were the positive and negativecontrols, respectively. Lymphocytes that had migrated to the lowerchamber were harvested by centrifugation and counted in ahematocytometer. Chemotaxis percentage was calculated as follows:(number of cells migrated to the lower chamber in the experimentalconditions−number of cells migrated in the negative control)/(number ofcells migrated in the positive control−number of cells migrated innegative control)×100. The results indicated that ADP-ribosyl HNP-1retained the ability to recruit T cells at a level that was comparableto unmodified HNP (FIG. 4C).

Example 9 Isolation of ADP-Ribosylated HNP-1 from Bronchoalveolar Lavage

To identify ADP-ribosyl HNP-1 in humans, bronchoalveolar lavage (BAL)was performed on 18 smokers and 10 non-smokers, none of whom had signsof pulmonary infection. Characteristics of the smokers are summarized inTable 1, below. The two populations did not differ significantly in age,gender, or pulmonary function. Samples (10 ml) of BAL from 18 cigarettesmokers (NHLBI IRB protocol #95-H-167) and 10 healthy non-smokers wereapplied to SepPack C-18 cartridges (Waters, Milford, Mass.) equilibratedin 10% isopropanol/0.1% trifluoroacetic acid (TFA), washed with the samebuffer, and eluted with 50% isopropanol/0.1% TFA. The eluted proteinswere concentrated in 6 M guanidine/0.1% acetic acid and separated on aVydac column CT8 HPLC column. Absorbence peaks (210 nm) corresponding tothe retention times of HNP-1 and ADP-ribosylated HNP-1 were analyzed byMatrix Assisted Laser Desorption Ionization-Time of Flight-MassSpectrometry (MALDI-MS). Molecules with masses corresponding toADP-ribosylated HNP-1 were further characterized. ADP-ribosylated HNP-1(0.2 μg) was incubated in 200 μl of 250 mM NaHCO₃, 25 mM MgCl₂, alkalinephosphatase (5 μg) and pyrophosphatase (5 μg) (Sigma), for 30 minutes at37° C., before termination of the reaction with 6 M guanidine,separation of reaction products by RP-HPLC and analysis by MALDI-MS.ADP-ribosylated HNP-1 (0.42 μg) was incubated overnight at 37° C. withrecombinant human ADP-ribosylarginine hydrolase (1 μg) in 100 μl of 50mM Tris (pH 7.5), 5 mM dithiothrietol, 10 mM MgCl₂ (Moss et al. Proc.Natl. Acad. Sci. 82: 5603-5607, 1985), followed by desalting andconcentration using Zip-Tip_(C18) (Millipore), and analysis by MALDI-MS.

BAL findings are summarized in Table 1, below. The total number of cellsrecovered from BAL of smokers was 4-fold that recovered from controls(P<0.001), with a greater absolute number of neutrophils in the BALfluid samples of smokers. RP-HPLC analysis of BAL fluid samples from 18smokers revealed peaks with the same elution time as authentic HNP in 12of the samples; four of these twelve samples showed peaks with elutiontimes identical to that of ADP-ribosylated HNP-1 (FIG. 5A). MALDI massspectral (MS) analysis demonstrated a molecule with a mass of 3,983daltons, confirming the presence of ADP-ribosylated HNP-1 (calculatedmass equivalent to 3,983 daltons) (FIG. 5B-1). Defensins were notdetected in BAL fluids from the non-smoker group.

The ADP-ribosylated HNP-1 was further characterized by incubating itwith pyrophosphatase/phosphatase to remove the ADP moiety, producingribosyl-HNP, or with ADP-ribosylarginine hydrolase, which cleaves theribose-arginine linkage, releasing HNP. After incubation withpyrophosphatase and alkaline phosphatase, MALDI analysis of the peakseparated by RP-HPLC separation, showed a molecule of 3,575 daltons,consistent with ribosyl-HNP-1, produced by removal of ADP from theADP-ribose-HNP-1 (FIG. 5B-2) (calculated mass equivalent to 3,574daltons). The site of linkage of ADP-ribose to HNP was confirmed afterincubation with ADP-ribosylarginine hydrolase, which released a moleculeof 3,444 daltons consistent with HNP-1 (calculated 3,443 daltons) (FIG.5B-3).

TABLE 1 Characteristics of smokers and bronchoalveolar lavage fluidsamples Population BronchoAlveolar Lavage Number Smoking History^(A)FV-1%^(B) Total Cells^(C) PMN^(D) HNP-1 ADP-R HNP-1 1 40/d 30 yrs 138245 2.1 + + 2 30/d 11 yrs 73 126 1.8 + − 3 40/d 5 yrs 93 140 1.6 − − 410/d 37 yrs 113 55 1 + − 5 40/d 12 yrs 110 95 1 − − 6 20/d 29 yrs 109103 1.9 − − 7 20/d 25 yrs 126 166 3 + + 8 20/d 15 yrs 95 108 3.5 + − 920/d 20 yrs 96 169 2 + − 10 10/d 9 yrs 143 46 3 − − 11 40/d 16 yrs 115150 0.5 + − 12 20/d 36 yrs 130 140 1.3 − − 13 30/d 15 yrs 80 280 8 + −14 20/d 30 yrs 96 135 0.8 + − 15 20/d 10 yrs 109 105 1 + − 16 30/d 26yrs 97 119 0.5 + + 17 30/d 30 yrs 66 44 0.5 + + 18 15/d 15 yrs 103 520.5 − − ^(A)cigarette per day, duration ^(B)percent of predicted FV-1(FV-1, Forced Expiratory Volume in 1 second) ^(C)total cells ×10⁶^(D)percent of total cells

Example 10 ADP-Ribosylation of Lysozyme by ART-1

ART-1 was collected from NMU (rat mammary carcinoma) cells, transfectedwith mouse ART-1 cDNA inserted in a pMAMneo plasmid. Protein expressionwas induced in the NMU cells with 1 mM dexamethazone for 24 hours. Cellswere then incubated with phosphatidylinositol-specific phospholipase C(PIPLC) (0.05 units per 300 μl Dulbeccos's phosphate buffered saline)for 1 hour at 37° C., sedimented by centrifugation and the supernatantcontaining ART-1 proteins was collected.

Lysozyme (5 μg, obtained from chicken egg white; Sigma, St Louis, Mo.)was incubated with mouse ADP-ribosyltransferase 1 (ART-1) in a reactionmix containing 50 mM potassium phosphate pH 7.5, 1 mM adenosinediphosphate ribose, 10 μM [³²P]NAD (10 μCi per assay reaction) for 1hour at 30° C. After TCA precipitation (10% final concentration),proteins were separated by SDS PAGE using a 12% acrylamide gel. The gelwas stained with Coomassie blue protein dye and exposed to X-ray film(Kodak XAR) for 5 hours. The resulting autoradiogram revealed a strongradioactive band, at approximately 14 kDa (molecular weight of lysozymeis approximately 14 kDa) in the lane corresponding to the [³²P]NADreaction mixture containing both ART-1 and 5 μg of lysozyme. The lanescontaining lysozyme, either with or without the [³²P]NAD reaction mix,were blank. Thus, the results indicate that ART-1 can ADP-ribosylatelysozyme.

This disclosure provides modified antimicrobial agents, such as modifieddefensin polypeptides. The disclosure further provides method ofmodulating an immune response using these modified polypeptides. It willbe apparent that the precise details of the methods described may bevaried or modified without departing from the spirit of the describedinvention. We claim all such modifications and variations that fallwithin the scope and spirit of the claims below.

1. A method for altering an antimicrobial activity of an isolated alphadefensin polypeptide, comprising: ribosylating or ADP-ribosylating atleast one arginine residue of the isolated alpha defensin polypeptidethat alters the antimicrobial activity of the polypeptide, wherein thealpha defensin polypeptide is human neutrophil peptide (HNP)-1, HNP-2,or HNP-3, and wherein HNP-1 comprises an amino acid sequence having atleast 90% sequence identity to the amino acid sequence set forth as SEQID NO: 2, HNP-2 comprises an amino acid sequence having at least 90%sequence identity to the amino acid sequence set forth as SEQ ID NO: 3,or HNP-3 comprises an amino acid sequence having at least 90% sequenceidentity to the amino acid sequence set forth as SEQ ID NO: 4, therebyaltering the antimicrobial activity of the isolated alpha defensinpolypeptide.
 2. The method of claim 1, wherein altering theantimicrobial activity comprises (i) an increase in neutrophilrecruitment or T-cell chemotaxis activity of ribosylated orADP-ribosylated HNP-1, HNP-2, or HNP-3, compared to an unmodified HNP-1,HNP-2, or HNP-3, or (ii) a decrease in cell lysis activity ofribosylated or ADP-ribosylated HNP-1, HNP-2, or HNP-3, compared to theunmodified HNP-1, HNP-2, or HNP-3.
 3. The method of claim 2, whereinaltering the antimicrobial activity comprises at least a 50% increase inneutrophil recruitment or T-cell chemotaxis, as compared to theunmodified HNP-1, HNP-2, or HNP-3.
 4. The method of claim 3, whereinaltering the antimicrobial activity comprises at least a 100% increasein neutrophil recruitment or T-cell chemotaxis activity, compared to theunmodified HNP-1, HNP-2, or HNP-3.
 5. The method of claim 3, whereinneutrophil recruitment activity comprises interleukin-8 release fromcells.
 6. The method of claim 2, wherein altering the antimicrobialactivity comprises at least a 50% decrease in cell lysis activity, ascompared to the unmodified HNP-1, HNP-2, or HNP-3.
 7. The method ofclaim 6, wherein altering the antimicrobial activity comprises at leasta 90% decrease in cell lysis, compared to the unmodified HNP-1, HNP-2,or HNP-3.
 8. The method of claim 1, wherein the human alpha defensincomprises an amino acid sequence having at least 90% sequence identityto the amino acid sequence set forth as SEQ ID NO:
 2. 9. The method ofclaim 8, wherein the human alpha defensin comprises an amino acidsequence having at least 95% sequence identity to the amino acidsequence set forth as SEQ ID NO:
 2. 10. The method of claim 1, whereinthe human alpha defensin comprises an amino acid sequence having atleast 90% sequence identity to the amino acid sequence set forth as SEQID NO:
 3. 11. The method of claim 10, wherein the human alpha defensincomprises an amino acid sequence having at least 95% sequence identityto the amino acid sequence set forth as SEQ ID NO:
 3. 12. The method ofclaim 1, wherein the human alpha defensin comprises an amino acidsequence having at least 90% sequence identity to the amino acidsequence set forth as SEQ ID NO:
 4. 13. The method of claim 12, whereinthe human alpha defensin comprises an amino acid sequence having atleast 95% sequence identity to the amino acid sequence set forth as SEQID NO:
 4. 14. The method of claim 1, wherein the human alpha defensincomprises the amino acid sequence set forth as SEQ ID NO:
 2. 15. Themethod of claim 14, wherein the human alpha defensin consists of theamino acid sequence set forth as SEQ ID NO:
 2. 16. The method of claim1, wherein the human alpha defensin comprises the amino acid sequenceset forth as SEQ ID NO:
 3. 17. The method of claim 16, wherein the humanalpha defensin consists of the amino acid sequence set forth as SEQ IDNO:
 3. 18. The method of claim 1, wherein the human alpha defensincomprises the amino acid sequence set forth as SEQ ID NO:
 4. 19. Themethod of claim 18, wherein the human alpha defensin consists of theamino acid sequence set forth as SEQ ID NO: 4.